Rapidly proliferating cancer cells are highly dependent on glutamine metabolism for the biosynthesis of protein, lipid, and nucleic acid building blocks, which are required for growth and proliferation. Many intermediates of these critical biosynthetic pathways are derived from the TCA cycle. Given that glucose-derived carbon is largely excreted as lactate rather than entering the TCA cycle (i.e., the Warburg effect), cancer cells adapt by upregulating glutaminolysis, which involves the 2-step conversion of glutamine to ?-ketoglutarate, a TCA cycle intermediate. As such, activation of the metabolic enzyme glutaminase C (GAC), which catalyzes the first step of glutaminolysis, namely the hydrolysis of glutamine to glutamate, is vital for the growth and proliferation of these cells. Indeed, a small molecule GAC inhibitor, referred to as 968, significantly reduces the growth of breast cancer cells. Furthermore, 968 does not affect the survival of nontransformed mammary epithelial cells, indicating that GAC participates in an alternative metabolic pathway that is dispensable for the growth of quiescent cells. Understanding the regulation of GAC is thus key for the design of novel chemotherapeutic agents that specifically target cancer cells without inducing the side effects often observed with classical chemotherapy. GAC activation has previously been linked to the signaling of hyper-activated Rho GTPases and the transcription factor NF-?B, but very little is known regarding the regulatory events controlling GAC activity. Thus, the specific aims of this proposal will identify the mechanisms leading to the upregulation of GAC activity in cancer cells. Preliminary data indicate that post-translational modifications (PTMs), particularly phosphorylation and the recently discovered lysine succinylation/malonylation, may be critical for the activation of GAC. Specifically, alkaline phosphatase treatment of GAC immunoprecipitated from transformed cells drastically reduces the basal activity of this enzyme, indicating that phosphorylation of GAC or a GAC-binding partner is required for high activity. On the other hand, knockdown of Sirtuin 5, a novel desuccinylase/demalonylase, reduces GAC basal activity by ~50%, suggesting that succinylation/ malonylation suppresses GAC activity. As such, mass spectrometry analysis will be used to identify GAC PTMs, followed by a mutation-based analysis in which phosphomimetic and phosphorylation- and succinylation/malonylation-defective mutants will be generated. These mutants will be examined for their ability to contribute to cellular transformation and to rescue the growth of GAC-depleted breast cancer cells. Additionally, preliminary results indicate that the splice-variant of GAC, termed kidney-type glutaminase (KGA), plays a role in regulating GAC activity through protein expression levels. As a long-term goal, the ability of KGA to contribute to transformation and regulate GAC activity will be investigated.